About this book

Michael Titheradge provides both the experienced researcher and the newcomer with time-tested techniques to cover all aspects of NO synthase and metabolism. Topics range from the cloning and expression of the different isoforms of NO synthase to the role of NO in DNA damage and apoptosis and include indirect and direct measurements of NO production, measurement on NO synthase mRNA both in situ and in vitro, and Western blotting and immunohistochemical localization of different isoforms of NO synthase. A variety of alternative techniques are described to enable researchers to set up an assay using either the simplest equipment or the most expensive EPR machines.

the protocols are concise and easy to follow. They are, in fact, extraordinary in their detail Nitric Oxide Protocols is a useful volume, and one that will certainly find its way into many laboratories entering the exciting field of nitric oxide research. - The Quarterly Review of Biology

Contents

Enzymology of Nitric Oxide Synthases. Purification of the Inducible Nitric Oxide Synthase. Purification of the Constitutive Nitric Oxide Synthase. Cloning and Expression of Human Inducible Nitric Oxide Synthase. Cloning and Expression of Human eNOS and nNOS Using the Baculovirus- Insect Cell System. Assay of NOS Activity by the Measurement of Conversion of Oxyhemoglobin to Methemoglobin by NO. Measurement of NOS Activity by Conversion of Radiolabeled Arginine to Citrulline Using Ion-Exchange Separation. Radiochemical Measurement of NOS Activity by Conversion of [14C]l-Arginine to Citrulline Using HPLC Separation. The Enzymatic Measurement of Nitrate and Nitrite. Determination of NOS Activity Using Cyclic-GMP Formation. Determination of NO with a Clark-Type Electrode. The Measurement of NO in Biological Systems Using Chemiluminescence. Measurement of NO Using Electron Paramagnetic Resonance. Measurement of eNOS and iNOS mRNA Expression Using Reverse Transcription Polymerase Chain Reaction. Measurement of NOS mRNA by Northern Blotting and the Ribonuclease-Protection Assay. Determination of NOS Isoforms by Western-Blot Analysis. Immunohistochemical Localization of NOS Isoforms. A Practical Protocol for the Demonstration of NOS Using In Situ Hybridization. Use of NO Donors in Biological Systems. Making and Working with Peroxynitrite. The Use of NO Gas in Biological Systems. A Microtiter-Plate Assay of Human NOS Isoforms. Use of Arginine Analogs as Inhibitors of Nitric Oxide Synthase in Rat-Aortic Rings. Measurement of Biopterin and the Use of Inhibitors of Biopterin. Biosynthesis. A Sensitive Assay for the Enzymatic Activity of GTP Cyclohydrolase. Simultaneous Measurement of Mitochondrial Function and NO. Release of NO from Donor Compounds: A Mathematical Model for Calculation of NO Concentrations in the Presence of Oxygen. The Determination of Nitrotyrosine Residues in Proteins. Measurement of DNA Damage Using the Comet Assay. Methods for the Study of NO-Induced Apoptosis in Cultured Cells. Index.

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